Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. DNA damaging agents and other genotoxins that arise from cellular metabolism, environmental sources or disease-related cellular defects contribute to cell death (e.g., neurodegeneration), gene mutations, gene rearrangements and in many cases, the onset of cancer, disease and aging phenotypes. In addition, many exogenous exposures such as chemotherapy and radiation treatment rely on the induction of tumor cell genotoxicity to mediate therapeutic response. Further, the ability to effectively and accurately repair spontaneous or induced DNA damage depends on the cellular DNA repair capacity. Therefore, the ability to quantify DNA damage and the rate of repair of the damage to the nuclear genome directly in human cells is critical in applications ranging from epidemiology to drug development. To address this technological need in the research community, to be better positioned to characterize the genotoxicity of newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair gene defects, we propose the development of the next generation in DNA damage detection and quantification technology. This proposal, to develop the 'DNA Repair on a Chip'technology, combines the use of agarose-based Microwell arrays, spatially- encoded cellular recognition, human tumor cell lines with genetically-defined DNA repair status and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays that will function as a tool to quantify DNA damage and measure cellular DNA Repair capacity at baseline and following genotoxin exposure on a single array or chip (DNA Repair on a Chip). The studies described in Aim 1 involve the development of a series of 24-well Spatially Encoded Microwell Arrays, with Microwells ranging from 10-50 5M in diameter and 20-50 5M in depth, suitable for gravity capture of a single cell of various sizes. Efficacy of the Microwell Arrays will be validated using radiation and small molecule inhibitors. Further, the sensitivity of the Microwell Arrays for analysis of cellular DNA Repair capacity will be evaluated using an isogenic panel of human tumor cell lines with defined defects in DNA Repair gene expression and following genotoxic stress. Iterative analysis and Microwell characterization will inform to finalize a set of 24-well Microwell Arrays for production and distribution. The studies described in Aim 2 involve additives to the Microwell Arrays that will enhance cell growth and attachment, providing optimal analysis of baseline DNA damage and most importantly, critical data on cellular capacity for in vivo repair post-damage. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses. Phase II of the project will be expanded to offer 96-well capability, end-user software for spatial recognition and quantitation plus micro-well additive options for specialized cell growth and attachment. PUBLIC HEALTH RELEVANCE: We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.